Parathyroid hormone-related peptide (PTH-rP) is the causative factor not only in humoral hypercalcemia of malignancy (HHM), but also in the local osteolysis associated with metastatic breast cancer. The reasons for overexpression of PTH-rP by cancer cells in these situations are unknown. In breast cancer, there is almost universal expression of PTH-rP in bone metastatic sites. Pharmacologic inhibition of PTH-rP transcription in bone causes reduction in osteolysis and tumor burden in that site (Gallwitz et al., 2002). Understanding the mechanisms responsible for PTH-rP overexpression by cancer cells could thus lead to identification of molecular targets for drugs that could be effective in the prevention or treatment of breast cancer and other cancers that are associated with increased PTH-rP expression. We hypothesize that the Gli family of transcription factors, the hedgehog signaling molecules in vertebrate cells, are important heretofore uninvestigated regulators of PTH-rP expression in metastatic human breast cancer cells. We propose that Gli family members have distinct and separate functions with respect to regulating PTH-rP expression. Gli2 is a powerful transcriptional activator, and the truncated form of Gli3 (produced by the actions of the specific E3 ubiquitin ligase beta-TrCP and subsequent proteasomal processing) is a strong repressor of PTH-rP transcription. (Gli1 and Gli2 are not processed to truncated repressor forms, and full length Gli1 and Gli3 have no significant effects on PTH-rP transcription according to our Preliminary Data). Our preliminary data suggests that human breast cancer cells overexpress Gli2, and that transient transfection of these cells with Gli2 and truncated Gli3 regulate PTH-rP expression. Expression of endogenous Gli2 in breast cancer cells correlates with PTH-rP expression and capacity to cause hypercalcemia or osteolysis. Cancer cells stably transfected with Gli2 cause enhanced osteolysis in vivo. We plan to test our hypothesis by determining if Gli2 and truncated Gli3 enhance or reduce PTH-rP expression and osteolysis induced by human breast cancer cells in vivo, identify the molecular mechanisms whereby Gli2 enhances PTH-rP transcription, determine the effects of loss of function of Gli2 and the E3 ligase for Gli3, beta-TrCP, on PTH-rP expression and osteolysis, and the role of Gli2 and truncated Gli3 in other PTH-rP overexpressing cancer cells that cause HHM. We propose therefore that the Gli family of transcriptional regulators represents an important molecular pathway that regulates PTH-rP expression in breast cancer cells, and subsequent breast cancer-mediated osteolysis.